The mechanical properties of tissues and cells (elastic modulus, deformability, or stiffness) are essential for determining the normal structure and function of tissues. Podocytes should have characteristic mechanical properties that permit them to maintain the structure and the integrity of glomerular capillary walls in the presence of capillary hemodynamic forces. The foot processes that form the slit diaphragms and the secondary processes that give rise to them contain actin bundles and actin cross-linking and bundling proteins (1-actinin and filamin) that determine their mechanical properties. Human glomerular diseases and a number of disease models that result in focal sclerosis result from mutations or abnormalities in podocyte cytoskeletal or adhesion proteins that should affect their mechanical properties. Our preliminary data show that in four models, glomeruli and podocytes are softer than normal. Our hypothesis is that podocytes have specific mechanical characteristics determined by the structure and composition of their cytoskeletons and their mechanical environment that permit them to support glomerular capillary structure and function. We focus on three mouse models, Col4a3-/- (Alport model, abnormal GBM), conditional podocyte integrin 21-/- (abnormal cytoskeletal-GBM connection), and Actn4-/- (abnormal actin cross-linking), that based on their distinct molecular pathology, will define specific mechanosensing and response pathways that lead to glomerular injury due to failure to sense or respond appropriately to mechanical signals. The three specific aims are: Aim 1. Define the mechanical properties (deformability) of mouse glomeruli and glomerular capillary walls from WT, Col4a3-/-, 21 integrin-/-, and Actn4-/- mice at a pre-disease state, and through the course of disease. Aim 2. Define the mechanical properties of mouse podocytes from WT, Col4a3-/-, 21 integrin-/-, and Actn4-/- mice, and the effects of different mechanical environments on them. Aim 3. Define the mechanisms by which 21 integrin and the cytoskeletal cross-linkers 1-actinin-4 and filamin, determine structural and biophysical responses to matrix-generated mechanical signals in podocytes to test for proteins or constructs that suppress the abnormal mechanical phenotype of the disease models. Approaching glomerular disease from this novel biophysical perspective, and defining the determinants of the mechanical properties of glomeruli and podocytes, will permit development of new lines of investigation and new interventions in glomerular diseases. PUBLIC HEALTH RELEVANCE: The part of the kidney that filters the blood (glomerulus) and is exposed to blood pressure and flow is damaged in many kidney diseases. In some cases the damage occurs due to defects in filter, and in others the damage occurs due excessive force from blood pressure and flow. In either situation, the mechanical properties of the filters are important. Most work in the field of kidney disease has focused on genetic or biochemical causes. We believe that as is the case for other tissues, mechanical factors in the tissue and environment are important components of normal function and disease. In this proposal, we will assess the mechanical properties of the filters and one important cell type in three different genetic disease models. Each disease model has a cause, the analysis of which with the mechanical properties of the disease model, will tell us a great deal about how the filters and cells we are interested in sense and respond to mechanical force. This new approach to kidney disease will give us information that will help us understand the causes of some kidney diseases, and may identify new approaches to their treatment, possibly by understanding how to strengthen the filters.